The invention discloses an amino acid detection method based on an aerolysin nanochannel. The method comprises the steps as follows: (1) designing DNA probes connected with formyl groups; (2) connecting amino acid to the DNA probes; (3) preparing the aerolysin bio-nanochannel: I activating aerolysin; II preparing a phospholipid n-decane solution; III preparing a phospholipid bilayer; IV forming the aerolysin bio-nanochannel; (4) detecting amino acid with aerolysin: I acquiring DNA probe single-molecular signals; II acquiring amino acid single-molecular signals; III acquiring DNA probe-amino acid single-molecular signals; IV counting the acquired DNA probe-amino acid single-molecular signals to obtain amino acid characteristic information. Accordingly, different amino acids can be detectedsimultaneously. The method can detect different amino acids simultaneously at the single molecule level, is convenient to operate, real-time and accurate, can realize ultrasensitive resolution and hasvery active significance in life science research.